Recently, studies have shown that human skin fibroblasts can be reprogrammed into induced pluripotent stem (iPS) cells via transduction of four viral genes (Takahashi, et al. (2007). Cell. 131: 861-872; Yu, et al. Science. 318 (5858): 1917-1920; Park, et al. Nature. 451: 141-146). These iPS cells have stem cell characteristics and can differentiate into cells of all three germ layers, a property known as pluripotency. The pluripotency concept has met skepticism regarding the abilities and potential dangers of these cells, however. Thus, the therapeutic uses of patient-specific iPS cells will be met with caution, as genetic alterations due to retroviral introduction must be investigated.
Creation of pluripotent stem cells from adult mouse cells without gene transfer has recently been reported in MedPage Today (John Gever, Edited, Published: Apr. 24, 2009.). The report based on Zhou H, et al.'s investigation demonstrate adult mouse cells can be reprogrammed into pluripotent stem cells with recombinant transcript factor proteins instead of genes (see Zhou H, et al. “Generation of induced pluripotent stem cells using recombinant proteins” Cell Stem Cell 2009; DOI: 10.1016/j.stem.2009.04.005). The transformed cells were able to form embryoid bodies and to differentiate into cells characteristic of the three primary germ layers: endoderm, mesoderm, and ectoderm.
A study by De Coppi et al. (Nat Biotechnol. 25(1):100-106 (2007) has shown that amniotic fluid cultures harbor multipotent stem cells that are not tumorigenic in mice. The researchers also found that amniotic fluid cultures initially show low levels (1%) of multipotent cells (CD117+ cells), a stem cell marker. After isolating such cells with microbeads, they were capable of differentiating into cells of any of the three germ layers.
De Coppi et al. isolated human and rodent amniotic fluid-derived stem (AFS) cells that express stem cell markers using immunoselection with magnetic microspheres from human amniocentesis specimens. AFS cells were grown in α-Minimum Essential Medium (α-MEM) containing 15% ES-FBS, 1% glutamine and 1% penicillin/streptomycin (Gibco), supplemented with 18% Chang B and 2% Chang C media (Irvine Scientific) at 37° C. with 5% CO2. Chang's media as supplemented in AFS cell cultures has been known for reduction of sera requirements in amniotic fluid cell culture. Chang and Jones reported (Prenat Diagn. 1985 September-October; 5(5):305-12) that addition of 10 growth promoting factors reduce serum requirement in the medium and the supplemented medium preserved the cells. However, it is not known if the supplemental growth factors preserved the cells for repeated passages.
In an earlier publication, Chang et al. (Proc Natl Acad Sci USA, 1982 August; 79(15):4795-9) also reported stability of human amniotic fluid cells grown in a hormone-supplemented medium. Chang et al. described development of a new supplemented medium to improve human amniotic fluid cell growth and to reduce the dependence on exogenously added serum. The Chang's medium includes a mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with Hepes, antibiotics, and 10 growth-promoting factors at 4% fetal bovine serum (see Chang et al. Table 1). Chang media compositions (CHANG MEDIUM®, Irvine Scientific), show Chang C medium formula contains an amount of Steroid Hormones. However, it is not known if the growth factors in the CHANG MEDIUM® play any role in the propagation of multipotent cells in amniotic fluid media.
Kim et al. (Cell Prolif. 40: 75-90 (2007)) reported isolation of fibroblastoid-type cells from human amniotic fluid (HAF) and subculture in culture medium containing Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, N.Y.) supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco), 3.7 mg/ml sodium bicarbonate, 10 ng/ml epidermal growth factor (EGF) (Peprotech, Princeton, N.J.) 10% fetal bovine serum (FBS) (Gibco). Seven days after the initiation of the culture, the medium was replaced with fresh ones, and subsequently replaced twice a week. The HAF-derived fibroblastoid-type cells that were passaged 8 times contained stem cells and were used for differentiation experiments
Crigler et al. (FASEB J. 21(9): 2050-2063 (2007)) demonstrated the presence of low numbers of multipotent cells (CD117+ cells) in the murine dermis and suggested that the cells can be repeatedly isolated from neonatal murine dermis by a sequence of differential centrifugation and be used for epidermal differentiation.
Recently Motohashi et al. reported that melanoblasts cells isolated from murine skin have multipotency and self-renewal capabilities. Isolated melanoblasts cells from mice skin were differentiated into neurons, glial cells, smooth muscle cells and melanocytes. Differentiation of the cells was inhibited by antagonist ACK2 (Stem Cells. April 2009, 27(4):888-97).
Stem cells harvested from a woman's bone marrow has been used to populate a stripped-down section of windpipe received from a donor and successfully transplanted into the woman's body (see NewScientist, “Woman receives windpipe built from her stem cells”, Nov. 19, 2008 by Andy Coghlan.
Stem cells derived from human menstrual blood have has been reported to prevent limbs with restricted blood flow from withering in mice (see NewScientist, “Stem cells from menstrual blood save limbs”, Aug. 19, 2008 by Alison Motluk. Investigators also believe that cells coming out of menstrual blood are regenerative (see Murphy et al., Journal of Translational Medicine, 6:45, Aug. 19, 2008).
Stevens et al. (see Stevens et al. Lab Invest. December; 84(12):1603-9, 2004) reported that fetal cells developed during pregnancy can persist in the mother's blood and tissues for decades. Studies have found that circulating stem cells can lead to liver regeneration with donor-derived hepatocytes. More specifically, male cells were found in livers of mothers who carried male babies, and these cells expressed hepatocyte antigens. This study provides a natural basis for regeneration of an organ from stem cells. Stevens et al. did not address if multipotent stem cells in human skin fibroblast samples can be propagated, differentiated and be used for regeneration of a desired organ, however. Rather, Stevens merely documented a natural phenomenon.
Umbilical cord blood is known to contain stem cells, and cord blood banks have been established.
There remains a need, however, to provide approaches for obtaining stem cells of both sexes without the need for recombinant gene or protein transfer in order to permit autologous therapy.
The present invention, provides for the first time methods for propagating, without the need for an initial isolation, multipotent stem cells from human skin fibroblast samples of both sexes of all races (including African-American and Caucasian female and male sources), using an appropriate medium, such as an amniotic fluid medium (AFM) and other media and various growth factors disclosed herein, and subsequent differentiation into cells of any of the three germ layers. It was surprising and unexpected that a rare cell type like a multipotent cell could be grown without first isolating because it was thought that other cell types, including non-potent fibroblasts, would overwhelm the rare cells types in culturing, particularly during multiple passages. The methods disclosed herein also allow for enhanced production of such multipotent stem cells without the need for gene or viral transduction of cell. Due to the efficiency of the disclosed methods, stem cells from an individual can be obtained and propagated to allow for autologous or otherwise type matched stem cell therapies, including tissue and organ grafts and supplementation, tissue and organ regeneration, and tissue and organ replacement. Prior to the present invention, such was simply not practical for the general public in a therapeutic setting. The invention also provides model systems to assess gene pathways in vitro and their effects in and during cell differentiation.